Cell-associated antigens, more specifically those with single or multiple transmembrane domains, are difficult to purify in their native conformation. In order to identify antibodies (or antibody fragments such as Nanobodies) against native epitopes which are able to modify the function of the target in vivo, it is crucial to administer the target antigen in its native conformation to the camelid [Dormitz et al. (2008). Trends in Biotechnology 26: 659-667]. In absence of purified native protein of these cell-associated antigens, the most applied immunization strategy consists of repetitive injections of whole cells functionally expressing the antigen of choice at regular intervals. Examples of targets for which such immunization strategy has been executed successfully (i.e. resulting in the identification of neutralizing, in vivo matured Nanobodies) are described in WO 05/044858 and WO 07/042,289). Repeated booster injections of target expressing cells, however, often result in diluted or non-detectable target specific immune responses, especially when the expression level of the target is low and the host cell background is highly immunogenic. The humoral response can be focused more towards the target by using a cell line of camelid origin, which is less immunogenic to llama. Nevertheless, repeated injections of (quasi)self-surface markers also result in a response against the camelid cell line surface markers.
The identification of (neutralizing) selective antibodies against GPCRs, Ion channels or any other type of multispanning cell surface marker is challenging [Michel et al. (2009). Naunyn-Schmied Archives Pharmacology 379:385-388], since i) most often no native protein is available for immunization or subsequent antibody identification, ii) multispanners often show low immunogenicity (due to a limited number of extracellular surface exposed amino acid residues compared to most single transmembrane receptors) and iii) multispanning surface molecules are often expressed at low densities.